Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.
Animals ; Rats ; Bacteremia/metabolism* ; Blood-Brain Barrier/microbiology* ; Caveolin 1/metabolism* ; Gingipain Cysteine Endopeptidases/metabolism* ; Permeability ; Porphyromonas gingivalis/pathogenicity* ; Transcytosis ; Virulence Factors/metabolism*

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
Animals ; Colitis, Ulcerative/microbiology* ; Mice ; Mice, Inbred C57BL ; Porphyromonas gingivalis/pathogenicity* ; Protein-Arginine Deiminases ; Virulence Factors

ABSTRACT The oral microbiome comprises several hundreds of bacterial species that contribute to periodontitis, the most complex polymicrobial inflammatory disorder. Porphyromonas gingivalis is a prominent periodontitis pathogen that produces gingipains as a major virulent factor. Gingipain facilitates P. gingivalis survival, pathogenicity, and growth. Several genes were identified to have a role in the regulating of P. gingivalis pathogenesis. Studies suggest that gingipains inhibition is key for the successful treatment of periodontitis. As of now, several gingipain inhibitors have been developed, some exhibit high inhibition activity against gingipains. However, most inhibitors offer unknown toxicity and undesirable side effects. Hence, the development of highly potent and safe gingipain inhibitor is a major concern for periodontitis treatment. The present review highlights the connectivity between P. gingivalis, virulent factors, and its gene, periodontitis, and gingipain inhibitors. Development of gingipains inhibitors would not only treat periodontitis but would also assist in the treatment of other associated systemic diseases, for example: rheumatoid arthritis, cardiovascular diseases, diabetes, and Alzheimer's disease.
Porphyromonas gingivalis--pathogenicity ; Gingipain Cysteine Endopeptidases

OBJECTIVE: Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. METHODS: THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05). CONCLUSIONS The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.
Adult ; Humans ; Lipopolysaccharides ; Macrophages ; metabolism ; Myeloid Cells ; Periodontitis ; metabolism ; microbiology ; Porphyromonas gingivalis ; pathogenicity ; Triggering Receptor Expressed on Myeloid Cells-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Fimbriae Proteins ; genetics ; Genotype ; Humans ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Virulence

OBJECTIVEThis aimed to investigate the effect of specific sequence oligodeoxynucleotide MT01 on the biological properties of osteoblasts invaded by Porphyromonas gingivalis (P. gingivalis ) by evaluating proliferation, cell cycle, and apoptosis.

METHODSMG63 osteoblasts were recovered and incubated with MT01, CpG ODN, metronidazole (MNZ), and gentamicin (GEN) for 3 h. P. gingivalis (the multiplicity of infection was 100:1) was added subsequently and cocultured for another 24 and 48 h. Cells with PBS comprised the blank group, whereas cells with P. gingivalis comprised the negative controls. Six experimental groups were established: PBS group, P. gingivalis group, MT01+P. gingivalis group, CpG ODN+ P. gingivalis group, MNZ+P. gingivalis group, and GEN+P. gingivalis group. The proliferative ability was measured by methyl thiazolyl tetrazolium assay, and the percentages of apoptosis and cell cycle were examined by flow cytometry.

RESULTSCompared with the blank group, proliferation increased significantly in the MT01+P. gingivalis group (P < 0.05). The ratio of cells was lower at the G₁ phase and higher at the S phase in the MT01+P. gingivalis group compared with the results in the P. gingivalis group (P < 0.05). Early cell apoptosis in the MT01+P. gingivalis group was significantly lower than that in the P. gingivalis group (P < 0.05).

CONCLUSIONMT01 can promote the proliferation, reduce the ratio of the G₁phase, increase the ratio of the S phase, and inhibit the early apoptosis of osteoblasts invaded by P. gingivalis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gentamicins ; pharmacology ; Humans ; Metronidazole ; pharmacology ; Oligodeoxyribonucleotides ; pharmacology ; Osteoblasts ; cytology ; drug effects ; Porphyromonas gingivalis ; pathogenicity

OBJECTIVEThis study measures the glutaredoxin (Grx) gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The involvement of the Akt-signaling pathway is also determined.

METHODSEA-hy926 cells were pretreated with 1,000 ng · mL⁻¹ P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS (1,000 ng · mL⁻¹ LPS), and carmus- tine (BCNU) groups (1,000 ng · mL⁻¹ LPS, and the EA-hy926 cells were pretreated with 25 μmol · ml⁻¹ BCNU for 30 min).

RESULTSGene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant (P < 0.05). After 12 h, the protein levels of Grx and phosphorylated-Akt (p-Akt) significantly increased in the LPS group (P < 0.05), whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels (P < 0.05). Moreover, a slight difference was observed in the total Akt protein levels in the three groups (P > 0.05).

CONCLUSIONGrx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU (a Grx inhibitor), which indicated that Akt might act as a downstream regulator of Grx.

Endothelial Cells ; Glutaredoxins ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Oxidative Stress ; drug effects ; Phosphorylation ; Porphyromonas gingivalis ; pathogenicity ; Proto-Oncogene Proteins c-akt ; drug effects ; Signal Transduction ; drug effects ; Umbilical Veins

BACKGROUNDThe occurrence and development of aortic aneurysm (AA) are associated with infection. Some researchers have detected the DNA of periodontal pathogens in AA samples in certain populations. However, it has not been done in Chinese population. The objective of this study was to evaluate the prevalence of periodontal pathogens in oral tissue samples and aneurysm samples of AA patients.

METHODSEighty-nine subjects with AA and 59 subjects without AA were examined. Periodontal clinical parameters were evaluated. Unstimulated saliva and subgingival plaque samples were collected from all subjects. Twenty-six dissected AA samples were obtained. Evidence of eight periodontal pathogens including Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Tannerella forsythensis (Tf), Treponema denticola (Td), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), and Prevotella nigrescens (Pn) was ascertained in all samples by 16S rRNA-based polymerase chain reaction (PCR) assay.

RESULTSThe periodontal indexes including plaque index (PLI), probing depth (PD), bleeding index (BI), and clinical attachment loss (CAL), of the six Ramfjord index teeth were significantly higher in the AA group than those in the control group (P < 0.01). Eight periodontal pathogens in subgingival plaque samples were more frequently detected in the AA group than in control group. The difference in prevalence between the groups was significant for six (out of eight) periodontal pathogens assayed (Pg, Pi, Fn, Pn, Tf, and Td, P < 0.01). Additionally, all eight periodontal pathogens were more frequently detected in saliva samples of the AA group than in those of the control group, again with six (out of eight) (Pg, Pi, Fn, Cr, Tf, and Td) displaying significant differences in prevalence between the two groups (P < 0.01). Out of 26 aneurysm samples examined, Pg, Pi, Fn, Cr and Tf were detected in 6 (23.1%), 2 (7.7%), 3 (11.5%), 1 (3.8%), 2 (7.7%), respectively, and Aa, Pn, and Td were not detected in dissected aneurysm samples.

CONCLUSIONResults of this study suggested that periodontal infection is associated with the occurrence of AA.

Aged ; Aggregatibacter actinomycetemcomitans ; genetics ; pathogenicity ; Aortic Aneurysm ; microbiology ; Female ; Humans ; Male ; Middle Aged ; Periodontitis ; microbiology ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Prevotella intermedia ; genetics ; pathogenicity ; RNA, Ribosomal, 16S ; genetics ; Treponema denticola ; genetics ; pathogenicity

Animals ; Cardiovascular Diseases ; etiology ; metabolism ; Humans ; Inflammation ; metabolism ; Periodontitis ; complications ; metabolism ; microbiology ; Platelet Aggregation ; physiology ; Porphyromonas gingivalis ; pathogenicity ; RNA, Messenger ; metabolism ; Receptor, PAR-1 ; metabolism ; Receptor, PAR-2 ; genetics ; metabolism ; Receptors, Proteinase-Activated ; metabolism ; Receptors, Thrombin ; metabolism

OBJECTIVETo evaluate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament cells (HPDLC) and the effect of Pg on bone resorption in periodontitis.

METHODSHPDLC were exposed to 25, 50 mg/L sonicated extracts of Pg for 6 h, HPDLC without treatment served as control. The expression of RANKL-OPG mRNA and protein were examined by real time polymerase chain reaction and Western blotting. OPG protein in the supernatant was examined by enzyme linked immunosorbent assay (ELISA). The data were statistically analyzed by SPSS 13.0 and one-way analysis of variance (ANOVA).

RESULTSWhen HPDLC were exposed to sonicated extracts of Pg, the expression of RANKL mRNA and protein in 25 mg/L and 50 mg/L groups were higher than that of control group (P < 0.05), the expression of OPG mRNA in 50 mg/L group (0.087 ± 0.021) was lower than that of control group (0.240 ± 0.019) (P < 0.05), and OPG protein in 25 mg/L and 50 mg/L groups (0.813 ± 0.007, 0.398 ± 0.009) was lower than that of control group (1.131 ± 0.005) (P < 0.01). OPG protein expression in the supernatant was not significantly different between experimental group and control group.

CONCLUSIONSSonicated extracts of Pg exposed to HPDLC can up-regulate RANKL expression, down-regulate OPG expression and influence bone metabolism.

Adult ; Cells, Cultured ; Humans ; Osteoprotegerin ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; Porphyromonas gingivalis ; pathogenicity ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sonication ; Young Adult